Transcriptional programs specify and elaborate cell identity during animal development, as a single cell gives rise to the hundreds of cell types that comprise the adult animal. The time, place, level and molecular context in which a gene is expressed are therefore critical for its function. Imaging techniques are currently in a unique position to capture all of these features simultaneously with sub-cellular resolution. In situ hybridization, where a target mRNA is hybridized to a complementary nucleic acid probe, is the primary method used to visualize mRNA expression in intact embryos and tissue samples. However, current detection methods for mRNA in situ hybridization do not take full advantage of the potential of imaging to capture quantitative information about the expression of many genes over space and time. Here, we propose to develop a fluorescent detection method for mRNA in situ hybridization based on the concept of a metafluorophore: a programmable DNA scaffold to target defined numbers of fluorophores to mRNA. Compared to current methods, this approach will improve signal to noise characteristics, will be quantitative, will be able to detecting many genes simultaneously and will be more feasible - in terms of cost, scale and time to usable data. PUBLIC HEALTH RELEVANCE: All cells in the body have the same genome, but acquire different capabilities by expressing subsets of their genes. This proposal develops technology to measure the level of expression of many genes in individual cells using fluorescent imaging, and applies it to understand how gene expression varies during development.